Open AccessExpression, purification, crystallization and preliminary X‐ray diffraction analysis of the TonB‐dependent haem outer membrane transporter ShuA from Shigella dysenteriae
Author(s) -
Brillet Karl,
Meksem Ahmed,
Thompson Andrew,
Cobessi David
Publication year - 2009
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309109008148
Subject(s) - shigella dysenteriae , crystallization , transporter , bacterial outer membrane , chemistry , membrane , biochemistry , microbiology and biotechnology , crystallography , biology , escherichia coli , gene , organic chemistry
As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram‐negative bacteria, the TonB‐dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His 6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X‐rays beyond 3.5 Å resolution were obtained by co‐crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X‐ray diffraction data at 2.3 Å resolution using one crystal of ShuA‐Pb, and at 3.2 Å resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA‐1 at SOLEIL.